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  In this study, we investigated the effect of nicotinic receptor agonists and antagonists on the analgesic response to morphine in the formalin test. In experiments conducted in mice, nicotine produced an early dose-dependent analgesic effect. At a dose of 0.5 mg/kg, mecamylamine, a nicotinic receptor inhibitor, suppressed the analgesic effect induced by 0.1 mg/kg nicotine in both stages of the formalin test, while hexamethonium had no such effect. Atropine, a muscarinic receptor antagonist, reduced the nicotine response at doses of 5 and 10 mg/kg. Mecamylamine, hexamethonium and atropine had no effect on morphine-induced analgesia. Administered separately, the antagonists had no effect on the analgesic response either. High doses of mecamylamine lead to an increase of the pain response. We conclude that cholinergic and opioid receptors play a possible role in the analgesic effect induced by nicotine.

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  There is evidence that sweeteners such as sucrose and saccharin interact with endogenous opioid systems. Further research has shown that feeding different concentrations of sucrose and saccharin alter latency in the tail-flick test. In this study, the influence of a 12-day regimen of different sweetening agents, sucrose (32%), saccharin (0.08%) and aspartame (0.16%) on morphine-induced analgesia in the formalin test was investigated. Male albino mice (20-27 g) were used for the experiments. The animals were given 12 days to adapt to the dietary conditions. An initial subcutaneous injection of saline or morphine (1.5, 3, 6 or 9 mg/kg) was given 30 minutes before the observation period. Recording of the early phase began immediately and continued for 10 minutes. Recording of the late response began 20 minutes after injection and continued for 10 minutes. Sucrose and aspartame increased the analgesia of morphine in the early phase while saccharin had no effect. On the other hand saccharin and sucrose decreased the effect of morphine in the late phase while aspartame increased the effect of morphine-induced analgesia. In conclusion, the present data provide further evidence for an important role of dietary variables in determining the effects of exogenous opioids on pain sensitivity.

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Amitriptyline, a tricyclic antidepressant agent is used as one of the analgesic drugs in different kinds of pain. In the present study the effect of local (subcutaneous) injection of amitriptyline (50 and 100 µg) on the acute and chronic pain using formalin test, was investigated. Our data show that local injection of amitriptyline to the paw receiving formalin, causes a decrease in pain in both phasic and tonic phases of formalin test. On the other hand, in the group that this drug was injected (100 µg) to the contralateral paw, no significant change was observed in the pain score with respect to the control group. Therefore, it seems that the observed effect is due to the local action of amitriptyline and not as a result of its systemic effect. Considering the above-mentioned results, it is concluded that: 1) Amitriptyline acts like a local anesthetic in both acute and tonic phases of formalin test. 2) The mechanism of analgesia in the acute phase is probably caused by sodium channel blockade. 3) The analgesic effect of amitriptyline in the tonic phase of formalin test might be due to its antihistaminic and anti-inflammatory effects at the peripheral level, and also is due to the changes in the CNS plasticity which occurs during the acute phase.

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It has been reported that neurons in the VL PAG projecting to nucleus raphe magnus (NRM) utilize excitatory amino acids as neurotransmitter. This projection plays an important role in the descending pain modulatory system. In order to determine the role of this pathway in pain perception, male rats received unilateral injection of ibotenic acid stereotaxically. For lesioning VL PAG, unilateral injection of ibotenic acid (0.2 and 0.5 µl) (a specific neurotoxin for EAA-containing neurons) was made. After a week recovery period, the nociception was evaluated using formalin test for a period of 60 min. At the end of experiments, animals were perfused by formaldehyde 10%. Serial sections (80 µm) wee prepared using vibratome. The sections were then Nissl stained and examined to determine the lesion location. The results revealed a significant increase in the first phase of formalin test. However, in the second phase, only 0.5 µl of the neurotoxin caused a significant decrease in pain perception. It is concluded that NMDA receptors within the PAG are involved in the perception of pain as measured in the formalin test.

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Introduction: Ultra low dose (ULD) morphine induces hyperalgesia which is mediated by excitatory Gscoupled opioid receptors. This study was designed to investigate the development of tolerance to hyperalgesic effect of morphine. Also we attempt to seek possible similarity, in view of Gs proteins, between hyperalgesic effect of ULD and hyperalgesic effect after tolerance to HD. Method: Male Wistar rats weighing 180-220 g were used. All injections were given intra peritoneally. For tolerance induction animals received ULD or HD for 5 days and at the 6th day tail flick test was performed before and 30 min after morphine administration. Effect of pretreatment with ULD on analgesic tolerance was assessed by injection of ULD before HD in 5 consecutive days then TF record was done after HD injection on 6th day. Time interval between injections was 15 minutes. Cross tolerance assay was measured by recording the response to a specified dose in 6th day after 5 days treatment with another dose. Oseltamivir, as a GM1 ganglioside inhibitor, was used for inhibition of Gs signaling. . Results: Our results showed that: 1) tolerance was established after chronic injection of ultra low dose (ULD) of morphine. 2) Pre-treatment by ULD reduced tolerance to therapeutic dose of morphine. 3) Cross tolerance to analgesia was observed after chronic administration of ULD. 4) Combination therapy with oseltamivir blocked hyperalgesia reduced analgesic tolerance and attenuated the development of tolerance to hyperalgesic effect of morphine. Conclusion: The results showed the partially common mechanism for development of tolerance to hyperalgesic and analgesic effect of morphine. Signaling through Gs proteins seems to be a common pathway.

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Introduction: Different mechanisms are involved in stress induced analgesia (SIA) and hyperalgesia (SIH). Repeated stress induces development of tolerance to SIA. The role of HPA axis and Gs signaling pathway in these effects are investigated in the current study. Methods: Forced swim stress (5 min/day) in water (20±1 ºC) was employed to adult male Wistar rats (200-250 g). The nociceptive threshold was assessed using tail flick test. Adrenalectomized (ADX) rats were also subjected to stress tests. Oseltamivir was used to block Gs signaling pathway. Results: Stress produced analgesia for 1 h (p<0.001) and hyperalgesia during 3-24 h after its induction (p<0.05). Repeated administration of the stress caused tolerance development to SIA and increased SIH recorded at 24 h after each session (p<0.001). Oseltamivir couldn’t reverse the SIH. Dexamethasone produced hyperalgesia from 30 min (p<0.001) to 24 h after its administration (p<0.01). Repeated injection of dexamethasone increased the hyperalgesia recorded at 24 h after treatment (p<0.001). In ADX animals SIA continued for 24 h (p<0.01). Adrenalectomy attenuated the chronic stress-induced SIA tolerance and eliminated SIH. Conclusion: SIH is suggested to be related to adrenal activity which also has a role in SIA tolerance. Upper parts of HPA axis seems to be responsible for SIA. Oseltamivir could not reverse the SIH. Therefore, the Gs signaling pathway activation by opioid system may not be responsible for SIH.

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Nimodipine, an L-type calcium channel blocker, can induce analgesia. However, it is not clear that this analgesic effect is at the level of spinal or supraspinal pain pathway. In addition, it has been reported that the analgesic effect of nifedipine, another L-type calcium channel blocker is related to the HPA axis, but there is no report indicating the role of this axis in the analgesic effect of nimodipine. Methods: Analgesia was measured by tail-flick (TF) test involving spinal reflexes and by hot-plate (HP) requiring an intact central nervous system. Assays were done before and 15, 30, 60 and 120 min after drug administration in the intact, sham operated and adrenalectomized rats. To identify the interaction between nimodipine and HPA axis, plasma corticosterone level was measured using the radioimmunoassay. Results: Nimodipine significantly decreased the plasma corticosterone level, and showed significant antinociception in both tests. Adrenalectomy potentiated the analgesic effect of nimodipine which was reversed by corticosterone replacement. Furthermore, nimodipine analgesic effect in ADX rats was more potent in HP test (compared to TF test). Nimodipine, at mentioned doses, did not alter animal’s movement indices in activity monitoring test. Conclusion: Nimodipine involves both spinal and supraspinal sites to control thermal pain transmission in presence of adrenal gland. It seems that there is a mutual interaction between nimodipine and HPA axis, especially at supraspinal levels.

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Background: Previous studies have indicated that administration of saffron extract could induced reward and reduces morphine reward as investigated by place preference and behavioral sensitization in male and female mice. In the present study, the effects of water extract of Crocus sativus on the acquisition and expression of tolerance to morphine-induced hyperalgesia in female N-MRI mice (20-25 g) were investigated. Matherila and Methods: Tail Flick technique was implicated in the present study. Morphine tolerance achived by morphine (50 mg/kg twice daily) injections for three consecutive days. On the 4th day of the experiments, morphine tolerance was assesed in animals by injection of effective dose of morphine (10 mg/kg). The extract of the C. sativus was administered during (development of tolerance) or after induction of morphine tolerance (expression). Results: Results showed that administration of morphine (1, 5, 10 and 20 mg/kg), induced a significant analgesia in animals. Administration of the plant extract (1, 2.5, 5, 10, 50 and 100 mg/kg) also produced analgesia which was statistically significant in dose 10 mg/kg of the extract. Injection of the plant extract (1, 2.5 and 5 mg/kg) in the test day, 30 min before morphine (10 mg/kg) reduced the expression of morphine tolerance. Administration of the extract (1, 2.5 and 5 mg/kg) during the induction of morphine tolerance, have not any effect on the development of morphine tolerance . Conclusion: It could be concluded that injection of the extract of C sativus can inhibit the expression but can not altered the acquisition of morphine tolerance. In addition, the extract could induced analgesia by it-self.

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Introduction: Based on human studies, inequality and social injustice have adverse effects on the individual and community health. In this study, the effects of food intake inequality and social status changes on pain perception and immunological factors were investigated in Balb/C mice. Methods: The present study was conducted by implementing different social stresses including food deprivation, food intake inequality and unstable social status (cage-mate change every 3 days) in 48 female mice. Formalin test was performed and thereafter the viability of peritoneal macrophages and spleen lymphocytes was evaluated by MTT assay. Concentrations of proinflammatory cytokines including IL-6, IL- 1 and TNF-α were also measured. Results: Our results showed that the implementation of food deprivation and inequality induced significant changes in chronic phase of formalin test compared to the control group (P<0.05). Pain perception was considerably decreased and this decline in inequality exposed subjects was well above the isolated ones. However, unstable social situation did not affect pain perception. Moreover, cell viability of peritoneal macrophages decreased, while cell viability of spleen lymphocytes and proinflammatory cytokines concentrations were increased in the serum of all stressed animals in comparison with controls (P<0.05). Conclusion: These results revealed that although food deprivation and social inequality can induce analgesia, some socioeconomic situations like social instability does not affect pain perception. All of these situations decrease cell viability in macrophages but enhance cell viability in lymphocytes. It seems that a proinflammatory stress condition is involved in these situations.

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Introduction: The current investigations on Health Equity, primarily point to the harmful health consequences of being in a stressful social hierarchy. The repetitive nature of social conflicts seems to favor the induction of hyperalgesia or hypoalgesia both in rodents and humans, and it can also affect the immune system. In this study, the effects of changes in social status on pain perception as well as alterations of pro-inflammatory cytokines were investigated in Balb/C mice. Methods: By implementation of a sensory contact model in 22 male inbred mice (stress group) from 30 mice of the Balb/c strain and modeling of dominance/submissive relationship, each mouse was injected by 20 μl of formalin 2% and their pain behavior was scored, then serum concentrations of proinflammatory cytokines were measured in all mice. Results: Our results showed that subordinate mice in chronic phase of formalin test were hypoalgesic as compared to the control and dominant mice (P<0.05). On the other hand, dominant mice were hypoalgesic as regards to subordinate mice during acute phase of formalin test. IL-1 and IL-6 concentrations in serum of dominant and subordinate mice were well above the control group. Conclusion: These results revealed that despite similar increase of proinflammatory cytokines' level in dominant and subordinate subjects social status can differently affect pain perception.

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Introduction: Despite significant progress in understanding pain control mechanism, there are numerous questions about central nervous mechanisms underlying stress-induced analgesia. The rostral ventromedial medulla (RVM) in the brainstem integrates a variety of functions, including pain modulation and pain perception. In the present study, we investigated the effect of temporary inactivation of RVM on stress-induced analgesia. Methods: This study was performed using adult male Wistar rats (200-250 gr). Swim stress was induced using a cylinder filled with water (50 cm height, 20±1°C) in which the rats were kept for 3 min. For induction of pain, 50 μL of 2% formalin was injected subcutaneously in the hind paw. For temporary inactivation of RVM, 0.5 μL of 2% lidocaine was injected into RVM. Results: Injection of lidocaine into RVM, before inducing swim stress, potentiated the anti-nociceptive effects of swim stress in phase 1 and phase 2A. In phase 2B swim stresses increased nociceptive scores of formalin test so administration of lidocaine into RVM inhibited the effect of swim stress. Conclusion: The result of this study demonstrated that temporal inactivation of RVM by lidocaine potentiated stress-induced analgesia.

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Introduction: Topiramate is an anti-convulsant drug, which produces its effects via glutamate metabotropic receptors inhibition and/or GABA receptor excitation. In the present study, attempts were made to investigate the effects of topiramate on the tolerance to morphine-induced analgesia activity in male NMRI mice (20-30 g). Methods: Hot plate method was chosen for the study. First of all the analgesic effects of morphine and topiramate on mice were investigated. Then, the animals became tolerant to morphine (50 mg/kg twice daily for three consecutive days). Different doses of topiramate were administered to the animals 30 min before each morphine (50 mg/kg) injections (acquisition) during tolerance development or on the test day, 30 min before the experiment. Results: Subcutaneous morphine injection (10 mg/kg) induced analgesia. However, intraperitoneal administration of topiramate (0.5, 2.5 and 5 mg/kg) had no effect. In addition, topiramate (0.5, 2.5 and 5 mg/kg) did not affect morphine-induced analgesia. Administration of a single daily dose of morphine (50 mg/kg twice daily) for 3 days, induced tolerance. Injections of topiramate (0.5 and 2.5 mg/kg) had no effects on the acquisition of morphine tolerance. However, topiramate (0.5 and 2.5 mg/kg) enhanced the expression of morphine tolerance. The drug reduced the expression of morphine tolerance at the dose of 5 mg/kg. Conclusion: Topirmate showed a biphasic effect on the expression of tolerance to morphine-induced analgesia in lower and higher doses which may be due to glutamate and/or GABAergic mechanisms.

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Introduction: Regulators of G-protein signaling protein negatively control G protein -coupled receptor signaling duration by accelerating Gα subunit guanosine triphosphate hydrolysis. Since regulator of G-protein signaling4 has an important role in modulating morphine effects at the cellular level and the exact mechanism(s) of adrenalectomy-induced morphine sensitization have not been fully clarified, the present study was designed to determine the changes in the levels of RGS4 mRNA and protein in intact and adrenalectomized (ADX) morphine-treated rats. Materials and Methods: All experiments were carried out on male Wistar rats. The tail-flick test was used to assess the nociceptive threshold and corticosterone levels were measured by radioimmunoassay. The dorsal half of the lumbar spinal cord was assayed for the expression of RGS4 using semi-quantitative RT-PCR and immunoblotting. Results: Results showed that the anti-nociceptive effect of intrathecal morphine (5 μg) was significantly increased in ADX rats. The levels of RGS4 mRNA and protein in ADX rats were similar to those in intact animals. However, morphine could elicit a significant increase in both mRNA and protein levels of RGS4 following adrenalectomy. In contrast, the pattern of RGS4 gene expression did not show significant changes in the lumbar spinal cord of intact animals after morphine injection. Conclusion: Our results demonstrate that in the absence of corticosterone, morphine increases RGS4 through promoting its gene expression.